Geographic Distribution and Seasonality of Brown Dog Tick Lineages in the United States

Abstract Two lineages of brown dog ticks (Rhipicephalus sanguineus sensu lato (s.l.), Latreille [Acari: Ixodidae]) have been described in North America: temperate and tropical. To characterize the distribution of these lineages across this region and evaluate seasonal activity, a 12S rRNA mitochondrial gene fragment was sequenced from R. sanguineus s.l. collected from hundreds of dogs and cats from different locations across 25 of the 50 states from 2018 to 2021. Infestations with temperate lineage predominated (78.5%) and were identified on pets from 20 states, with most (83.5%) from areas with annual mean daily average temperature <20°C. Tropical lineage submissions were less common (19.3%), submitted from 15 states, and most (80.0%) tropical lineage ticks were from areas with an annual mean daily average temperature >20°C. Although travel history was not obtained for all dogs, when tropical lineage infestations were found in colder regions, follow up conversations with veterinarians suggested some of these infestations may have resulted from recent travel of dogs. A limited number (2.2%) of dogs from Arizona and Texas were co-infested with both lineages. Both temperate and tropical lineage ticks were collected from pets in every month of the year. Temperate lineage infestations were primarily collected March through August while tropical lineage infestations were more often collected June through November. These data confirm at least two lineages of R. sanguineus s.l. are present in the United States, each predominating in distinct, overlapping geographies, and suggest that peak activity of each lineage occurs at different times of the year.

between temperate and tropical lineage brown dog ticks are infertile, suggesting they are distinct, reproductively incompatible species (Szabó et al. 2000, Levin et al. 2012. A male brown dog tick of the temperate lineage was described as neotype, leading to assignment of the temperate lineage as Rhipicephalus sanguineus sensu stricto (s.s.) (Nava et al. 2018). More recently, the name Rhipicephalus linnaei, Audouin [Acari: Ixodidae] was proposed for the tropical lineage (Šlapeta et al. 2021). Both R. sanguineus s.s. (temperate lineage) and tropical lineage brown dog ticks have been documented in the United States (Zemstova et al. 2016, Jones et al. 2017, Brophy et al. 2022. Research in this area is ongoing and a consensus on species nomenclature for the tropical lineage in the Americas has not been reached. Accordingly, we use the terms R. sanguineus s.s. (temperate lineage) and tropical lineage in the present paper.
Limited data are available regarding the geographic distribution and seasonal activity of R. sanguineus s.l. in North America. However, tropical lineages are predominantly collected in regions where the annual mean daily average temperature is >20°C (e.g., Florida, Hawaii, and southern Texas along the Mexico border), while R. sanguineus s.s. (temperate) lineage is usually found in cooler, drier climates and thus thought to be more widely distributed on the continent (Eremeeva et al. 2011, Zemstova et al. 2016, Jones et al. 2017. Historic seasonality patterns were largely reported before awareness of distinct species within the complex and thus are best viewed as R. sanguineus s.l. of unknown lineage. Under laboratory conditions, the life cycle of R. sanguineus s.l. is most efficient at temperatures of 20-35°C and relative humidity of 35-95% (Koch 1982, Dantas-Torres 2010. Provided canine hosts are present, R. sanguineus s.l. activity may be sustained year-round indoors. In warmer climates these ticks also persist outdoors although oviposition, egg hatching, and molting of immature stages are less likely to occur in lower temperatures (Koch 1982, Dantas-Torres 2010. Phenology of the different lineages and response to abiotic climatic clues (e.g., temperature, humidity) appears to vary, affecting diapause and seasonality of activity (Labruna et al. 2017). The present study was conducted to more fully characterize the geographic distribution and begin to assess seasonality of the two predominant lineages of R. sanguineus s.l. active in the United States.

Materials and Methods
Rhipicephalus sanguineus s.l. used in this study were collected from dogs and cats through a national survey in the United States as previously described (Saleh et al. 2019). In total, 214 veterinary practices from all 50 states were enrolled in the study and provided materials for tick submission; submissions also were accepted from additional, nonenrolled practices. Briefly, invitations were sent to veterinary clinics in each state until 2-4 clinics had been enrolled in each state. All submissions were voluntary, and no attempt was made to normalize tick collection efforts across the country. In addition, we did not limit enrollment and thus some municipal areas were represented by multiple clinics while enrollment was sparse in other areas. Each enrolled practice was provided a tick submission kit consisting of instructions, submission forms, tick containers, forceps, and prepaid mailing envelopes. A study website also provided instructions and an email link was available to address any questions (Saleh et al. 2019). Veterinary staff were asked to remove all ticks identified on a dog or cat, regardless of species, and place the ticks in a hard-plastic container with a tightly fitting lid; occasionally tick submissions were made using serum tubes or other rigid, sealed containers. The container with ticks was then sealed in a plastic bag, a submission form completed, and both specimen and submission form shipped to our laboratory. Geographic location of the veterinary practice where ticks were collected (City, State) and date ticks were removed from the infested animal were recorded. A map of annual mean daily average temperatures was obtained from the United States Climate Atlas, National Centers for Environmental Information (NOAA 2022). Ticks received were identified morphologically to species and stage following standard keys (Clifford et al. 1961, USDA 1976 and then preserved in 70% ethanol at −20°C until further testing. Only ticks identified morphologically as R. sanguineus s.l. were included in the present study; other tick species were also received and are reported elsewhere (Saleh et al. 2019).
Total nucleic acid was extracted from R. sanguineus s.l. in each submission as previously described (Jones et al. 2017). Briefly, adult R. sanguineus s.l., or immature R. sanguineus s.l. when no adults were submitted, were dissected and all internal contents were removed and extracted with a commercial kit (illustra blood genomicPrep Mini Spin Kit, Cytiva, Piscataway, NJ; or QIAamp DNA Blood Kit, Qiagen, Germantown, MD) following the manufacturer's instructions. A 12S rRNA mitochondrial gene segment (340-370 bp) was amplified from each specimen as previously described (Szabó et al. 2005); positive amplicons were purified using a commercial kit (Wizard SV Gel and PCR Clean-Up System, Promega, Madison, WI) and sequenced using Sanger method at Oklahoma State University Molecular Core Facility (Stillwater, OK). Each sequence was compared to all available 12S rRNA gene sequences of R. sanguineus s.l. in GenBank and to a curated subgroup of reference sequences from confirmed lineages (KC243786-KC243807, OM985276-OM985391, OM177220, OM177221) with R. sanguineus s.l. lineage assigned when identity to reference sequences varied by <1% (Dantas-Torres et al. 2013, Brophy et al. 2022, Mumcuoglu et al. 2022. To determine if pets were co-infested with multiple lineages, sequence was obtained from up to 10 individual ticks submitted from a given pet. Confidence intervals (95%) of all proportions were calculated by modified Wald (Agresti and Coull 1998). Seasonality was evaluated with Chi square tests and a P value significance threshold of 0.05.

Hosts and Tick Lineages
All ticks included in the present study were confirmed to be R. sanguineus s.l. by morphology. Additional tick species were also submitted but were not included. A single R. sanguineus s.l. was available for sequencing from each of 154 pets (154/362; 42.5%; 95% CI 37.5-47.7), while more than one R. sanguineus s.l. (n = 2-10) was available for sequencing from each of 208 pets (208/362; 57.5%; 95% CI 52.3-62.4). Amplification and sequencing of a 12S rRNA gene fragment from 1 to 10 ticks from each pet (

Geographic Distribution of Collections
R. sanguineus s.l. were submitted from a total of 25 states; R. sanguineus s.s. (temperate lineage) ticks were submitted from 20 states and tropical lineage ticks were submitted from 15 states ( Fig. 1; Table 1). Mixed infestations of R. sanguineus s.s. (temperate lineage) and tropical lineage were identified on dogs in two states, including 4 dogs from the same household in Arizona, 3 additional dogs from Arizona, and 1 dog from Texas. Most (237/284; 83.5%; 95% CI 78.7-87.3) R. sanguineus s.s. (temperate lineage) infestations were from areas where the annual mean daily average temperature is <20°C although this lineage was also identified on several dogs at or near the edge of this temperature range in southern California, Arizona, and Texas (Fig. 1) (Fig. 1). When tropical lineage ticks were identified from an unexpected locale, an attempt was made to contact the submitting veterinarian to inquire about recent travel history of the dog; 5 confirmed the dog had recently traveled to a warmer region, 2 denied any recent travel, including one dog from northern Texas and one dog from Wisconsin, and the remaining 7 did not respond or were unable to obtain travel information from the client (data not shown).

Discussion
The initial recognition that R. sanguineus s.l. comprises multiple tick species with similar morphology inspired subsequent efforts from researchers on several continents to understand the identity and diversity of lineages of Rhipicephalus spp. infesting dogs in a given region (Szabó et al. 2005, Burlini et al. 2010, Moraes-Filho et al. 2011, Nava et al. 2012, Dantas-Torres et al. 2013, Liu et al. 2013, Dantas-Torres et al. 2017, Hornok et al. 2017, Chitimia-Dobler et al. 2017, Chitimia-Dobler et al. 2019, Chandra et al. 2021, Páez-Triana et al. 2021, Mumcuoglu et al. 2022). In the United States, at least two lineages of R. sanguineus s.l. have been described: R. sanguineus s.s. (temperate lineage) in Arizona, California, Oklahoma, and Texas, and tropical lineage (referred to as R. linnaei by some authors) in Arizona, Florida, Hawaii, and southern Texas, with sympatric populations of the two lineages in some areas of Arizona and Texas (Eremeeva et al. 2011, Zemstova et al. 2016, Jones et al. 2017, Brophy et al. 2022. Results from the present study suggest that the R. sanguineus s.s. (temperate lineage) has a wider geographic distribution in the United States than previously recognized, with this tick identified on dogs or cats in 20 different states, although travel of pets may also lead to short-lived introduction of brown dog ticks to some premises. In addition, R. sanguineus s.s. (temperate lineage) ticks predominated among R. sanguineus s.l. infestations submitted by veterinarians, responsible for over three-quarters of the brown dog tick infestations identified from dogs and all the infestations found on cats in the present study. However, because ticks were collected passively and submissions were not balanced geographically, the apparent predominance of R. sanguineus s.s. (temperate lineage) in the present study may not accurately reflect the actual prevalence of each lineage throughout the United States.
Infestations with the tropical lineage of R. sanguineus s.l. were comparatively less common in the present study, and, when identified, largely restricted to the southernmost areas of the United States. Lineages of R. sanguineus s.l. appear to segregate according to elevation, latitude, and climatic factors (Zemtsova et al. 2016, Jones et al. 2017, Hornok et al. 2017, Sánchez-Montes et al. 2021, Brophy et al. 2022. For example, the tropical lineage has been found to persist primarily in areas with a mean average daily temperature >20°C, a limitation often attributed to the poor survivability of fed immature stages in harsher winter conditions (Zemtsova et al. 2016, Jones et al. 2017, Labruna et al. 2017, Saleh et al. 2021) and, in warmer countries, tropical may be the only lineage identified (Jones et al. 2017, Páez-Triana et al. 2021, Chandra et al. 2021. Accordingly, over 80% of the tropical lineage infestations identified in the present study were found on dogs in the warmest regions of the United States including southern California and southern Nevada, where this lineage has not been previously reported. However, we were surprised to also identify tropical lineage R. sanguineus s.l. from dogs from states with much cooler climates, including far northern states like Michigan, Minnesota, Washington (state), and Wisconsin, and confirmed that at least some of these likely resulted from recent travel of dogs with their owners. Recent travel history should always be a consideration when interpreting data on tick distribution based on ticks removed from companion animals or people, particularly when ticks are found in an unexpected region or at an unusual time of the year (Jones et al. 2017, Saleh et al. 2019, Xu et al. 2019, Eisen and Paddock, 2021Brophy et al. 2022 Apparent differences in vector capacity between R. sanguineus s.s. (temperate lineage) and tropical lineages may be epidemiologically important and could influence where geographically infections with particular pathogens are more commonly diagnosed (Dantas-Torres and Otranto 2015). For example, experimental and epidemiologic data indicate that E. canis, causative agent of canine monocytic ehrlichiosis (CME), is primarily transmitted by tropical lineage R. sanguineus s.l. and thus CME cases are more commonly diagnosed in regions where the tropical lineage predominates (Beall et al. 2012, Moraes-Filho et al. 2015. Conversely, Ehrlichia chaffeensis and Ehrlichia ewingii, which share Amblyomma americanum as primary vector, more commonly infect dogs in states in the United States where R. sanguineus s.s. (temperate lineage) predominates and thus transmission of E. canis would be expected to be less common, although brown dog ticks (lineage not specified) have been implicated as a potential secondary vector of E. chaffeensis and E. ewingii in some regions (Ndip et al. 2007, Beall et al. 2012, Zemstova et al. 2016. Although more research is needed, some data suggest that other pathogens transmitted by R. sanguineus s.l., including Rickettsia rickettsii, Anaplasma platys, and Hepatozoon canis, may have similar affinity for transmission by only individual lineages within the R. sanguineus s.l. species complex (Sanogo et al. 2003, Eremeeva et al. 2011, Demoner 2013. The extent to which distinct lineages of R. sanguineus s.l. serve as important vectors for both canine and zoonotic disease agents warrants further investigation, particularly given the importance of brown dog tick transmitted R. rickettsii as a public health concern (Demma et al. 2005, Álvarez-Hernández et al. 2017. The present paper also provides the first report in North America of 12S rRNA sequence consistent with an R. sanguineus s.l. lineage originally referred to as Rhipicephalus sp. I on three different dogs, including two from New Mexico and one from Idaho. This lineage (Rhipicephalus sp. I or 'southeastern Europe lineage') was originally identified in Italy and Greece and has since been described from eastern Europe and the Middle East (Dantas-Torres et al. 2013, Chitimia-Dobler et al. 2017, Chitimia-Dobler et al. 2019, Mumcuoglu et al. 2022. Although Rhipicephalus sp. I has been shown experimentally to successfully breed with R. sanguineus s.s. (temperate lineage, also Rhipicephalus sp. II), and limited sympatric populations have been described, the two temperate lineages (R. sanguineus s.s. and 'southeastern Europe lineage' Rhipicephalus sp. I) appear both genetically and geographically distinct (Chitimia-Dobler et al. 2017, Chitimia-Dobler et al. 2019. Alternatively, our finding may be an artifact from detecting low frequency haplotypes in ticks with mitochondrial heteroplasmy . Two of the three dogs with 'southeastern Europe lineage' Rhipicephalus sp. I temperate lineage ticks in the present study were also co-infested with a greater number of R. sanguineus s.s. (temperate lineage) ticks.
Regardless, the finding of a small number of Rhipicephalus sp. I or 'southeastern Europe lineage' ticks on dogs in the United States in the present study was unexpected and may represent introduction following travel much like our discovery of tropical lineage ticks on dogs in unexpected geographies (Fig. 1). Careful monitoring of tick populations is important to detect introductions when they occur, including ticks introduced from other continents (Beard et al. 2018). Tick populations in North America, as in other areas of the world, are continuing to expand, bolstered in part by warmer temperatures that support establishment once introductions occur (Minigan et al. 2018, Sonenshine 2018. Our findings support the interpretation that sympatric populations of R. sanguineus s.s. (temperate lineage) and tropical lineage are present in Arizona, southern Texas, and southern California, as has been suggested by others, although further research that includes environmental collections and full travel history on all dogs would be needed to confirm this assertion (Jones et al. 2017, Brophy et al. 2022. Indeed, four of the eight dogs found co-infested with both R. sanguineus s.s. (temperate lineage) and tropical lineage ticks in the present study were from the same household. Given the endophilic propensity of the R. sanguineus s.l. complex, multiple lineages can persist in the same region (Dantas-Torres et al. 2008). To limit further geographic spread, all dogs should be routinely treated with acaricides and especially when traveling between regions; this recommendation is particularly important when owners travel with pets to tropical climates during the cooler months when ticks may not be a major concern at home ( Wright et al. 2020).
The seasonality of R. sanguineus s.l. in the United States is not well documented and largely based on historic work prior to recognition of distinct lineages (Koch 1982). Survivability likely depends on vulnerability of different stages to the elements in a given region although once indoor infestations of are established, R. sanguineus s.l. activity may be sustained year-round provided dogs are available as a food source (Dantas-Torres 2010). In warmer climates, R. sanguineus s.l. can also survive outdoors and are remarkably resistant to desiccation (Koch 1982, Dantas-Torres 2010. When outdoors, this species tends to seek refuge between rocks or in cracks and crevices in the ground and stony outcroppings; ovipositing females are found in these protective niches to ensure their safety and that of their progeny (Walker et al. 2000, Dantas-Torres 2010. Data from the present study suggests that, although both lineages were collected from pets throughout the year, R. sanguineus s.s. (temperate lineage) may be most active in spring and summer, when conditions in temperate areas are more hospitable, and that tropical lineage ticks are more likely to be found in late summer and fall. However, as has been noted in prior research, the passive sample collection strategy used limits drawing firm conclusions about seasonality (Brophy et al. 2022). If supported by subsequent, prospective studies of R. sanguineus s.l. activity, this difference may reflect the absence of a pronounced winter diapause in the tropical lineage and the presence of winter diapause in R. sanguineus s.s. (temperate lineage) (Labruna et al. 2017). Additional field work that carefully controls for recent introduction via travel from another region and consistent assessment of dogs throughout the year will be needed to confirm precise seasonal patterns. However, the present study indicates that both lineages are present on dogs year-round in the United States, supporting recommendations to maintain dogs on consistent tick control (Creevy et al. 2019).
One limitation of the present study was lack of complete sequence data for all ticks on heavily infested dogs. Previous research from our group did not reveal the presence of mixed R. sanguineus s.s. (temperate lineage) and tropical lineage infestations on dogs (Jones et al. 2017), but subsequent work by others in Arizona, USA, did occasionally detect R. sanguineus s.s. (temperate lineage) and tropical lineages from the same dog and premise (Brophy et al. 2022). Although most dogs in the present study were infested with only a single lineage of R. sanguineus s.l., sequencing multiple (n = up to 10) ticks from the same dog revealed occasional mixed infestations with both R. sanguineus s.s. (temperate lineage) and tropical lineage present, as well as diversity in 12S rRNA gene sequence of R. sanguineus s.s. (temperate lineage) as previously described (Jones et al. 2017, Brophy et al. 2022. Indeed, sequencing at least 5 ticks from each pet, when possible, revealed multiple 12S rRNA sequences of R. sanguineus s.s. (temperate lineage) in half of the infested pets in the present study (data not shown), supporting the interpretation that 12S rRNA sequence variation is common in this tick species. In addition, sequencing multiple ticks revealed Rhipicephalus sp. I, a southeastern Europe lineage, on two dogs that were also infested with R. sanguineus s.s. (temperate lineage) more commonly found in the United States (Dantas-Torres et al. 2013, Zemtsova et al. 2016, Jones et al. 2017, Brophy et al. 2022). Had we sequenced a larger number of ticks from these dogs, greater sequence diversity may have been identified.
Other limitations include the passive method of sample submission, an incomplete travel history for most dogs, and lack of information about acaricidal treatment of dogs. Passive or convenience sampling can result in under-recognition of infestations in general and may bias seasonality data if infestations are largely only identified during peak tick activity, thereby limiting documentation during other times of the year (Brophy et al. 2022). A complete recent travel history was not routinely collected and, when obtained, relied on owner recollection. We also did not have detailed records regarding contact with other dogs such as recent adoption from a shelter, dog day care attendance, or stay at a boarding kennel, factors which may increase both risk of infestation and propensity for mixed lineage infestations as R. sanguineus s.l. are acquired from environments with dogs (Dantas-Torres 2010). Treatment of dogs with acaricides, if any, was also not recorded and may have limited the number of R. sanguineus s.l. specimens available for sequencing from each infested pet.
Finally, because our goal was understanding biogeography and seasonality of the two main lineages rather than deep phylogenetic analysis, we chose to only sequence a 12S rRNA mitochondrial gene fragment in the present study. Research from other regions using ticks held in museum collections for over a century has shown that this marker (12S rRNA gene) is useful and stable over time and allows consistent identification of established lineages. Sequencing additional gene targets would not be expected to shift identification of tick lineage (i.e., R. sanguineus s.s. (temperate), tropical, or southeastern Europe Rhipicephalus sp. I), but such data would support more robust phylogenetic inquiry, particularly within lineages (Dantas-Torres 2010, Dantas-Torres et al. 2018, Chandra et al. 2021). In addition, sequence could not be successfully amplified from ticks from 16 dogs, likely due to nucleic acid degradation prior to submission; data from these infestations may have extended the known distribution and seasonal activity of the different lineages of R. sanguineus s.l. active in the United States. Nevertheless, the findings in the present study advance our knowledge of distribution of R. sanguineus s.s. (temperate lineage) and tropical lineage in the United States, suggest these two species may have different peak seasonal activity periods, and support the interpretation that, while uncommon, co-infestation with both lineages occasionally occurs, an important consideration for future research (Zemtsova et al. 2016, Jones et al. 2017, Brophy et al. 2022.